Development of Mechanism-Based PET Flow Tracer Abstract: Cardiovascular disease is among the leading causes of death in the United States. Myocardial perfusion imaging (MPI), a versatile tool in clinical diagnosis, plays an important role in the noninvasive assessment of coronary artery disease (CAD). Currently, common single-photon emission computed tomography (SPECT) MPI agents comprise 201Tl or moderately hydrophobic and cationic 99mTc-complexes, such as 99mTc-sestamibi and 99mTc-tetrofosmin, for determining myocardial blood flow (MBF) in patients. However, SPECT imaging agents have inherent limitations, including the continuing threat of serious shortages of 99mMo/99mTc-generators. Additionally, current SPECT tracers also suffer from shortcomings in pharmacokinetics, myocardial extraction, redistribution of the radiotracer to non-targeted tissues over time, and non-linearity of uptake at elevated blood flow (the roll-off phenomenon). By comparison, positron emission tomography (PET) provides technical advantages, including higher spatial resolution, improved attenuation correction, and the capability to perform quantitative measurements at the peak of stress. Commonly employed PET MPI tracers are: 82RbCl, 13NH3, and H215O. However, the utility of these agents is limited due to their short physical half-life (<10 min), thus posing difficulties for easy access to these agents. Other promising 18F-labeled agents such as18F- BnTP, a mitochondrial membrane potential probe, and 18F-BMS-747158, 18F-10, 18F-RP1004, and 18F-MCI27, mitochondrial complex I inhibitors, have various strengths and weaknesses, but also depend on a 18F-based distribution model, which may not be readily accessible to underserved regions of the U.S. or other developing countries. Thus, PET tracers demonstrating high myocardial first pass extraction, rapid clearance from the liver, retention within the myocardium to enable delayed imaging, and based on isotopes that could potentially be generator-produced on site (rather than cyclotron produced) would facilitate wide access to PET MPI. To address this unmet goal, we have identified a lead cationic and moderately hydrophobic radiopharmaceutical 1A based on generator-produced gallium-68 that demonstrates high extraction into the myocardium of normal mice and rats, including efficient clearance from the blood pool and liver driven by the multidrug resistance P- glycoprotein (Pgp) transporter. The agent 1A is retained in myocardium over time and remains nonmetabolized, allowing stable distribution and high count statistics that translate into high quality myocardial images. Importantly, both microPET/CT (68Ga) and nanoSPECT/CT (67Ga) reveal high accumulation of 1A in heart followed by facile clearance from liver 60 min post intravenous injection in rats. Finally, following intravenous injection of 67Ga-1A, nanoSPECT/CT imaging clearly visualized the non-perfused region of the left ventricle wall indicating the potential of our lead agent to noninvasively image the myocardial perfusion defect in rat models. Thus, armed with these pilot data, specific objectives of this R01 proposal are: 1) Perform a focused SAR study to further optimize biochemical targeting and pharmacokinetics of our lead 68Ga- radiopharmaceutical as a potential MPI agent. 2) Perform mechanism-based assessment and validation of novel 68Ga-radiopharmaceuticals derived from our focused SAR studies in cardiomyocytes, HEPG2 cells, Pgp- expressing KB cells, Pgp-transfected MCF-7 cells, and correlate these data with 99mTc-Sestamibi, a well- validated and FDA approved SPECT probe under similar conditions. 3) Evaluate promising leads via pharmacokinetic analysis in rats, determining extraction fractions of radiopharmaceuticals into myocardium and comparative analysis of heart/liver ratios under similar conditions with 99mTc-Sestamibi and other potential 18F MPI agents. 4) Characterize and validate the lead 68Ga-radiopharmaceutical 1A or other potential leads emerging through SAR studies as a noninvasive MPI agent by assessment of myocardial infarct size, first pass extraction, and flow-extraction correlation under stress/rest conditions in rats, followed by assessment of leads in rabbits, using microPET imaging. 5) Characterize the potential of our lead agent or a second-generation 68Ga- radiopharmaceutical as a MPI agent in canine models; perform safety pharmacological profile studies, dosimetry analysis, and prepare eIND filing materials. Successful execution of the outlined objectives should provide a molecular-targeted 68Ga-PET agent for MPI and management of CAD.